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O▪29 Microarray technology to assess gene expression profiles of human oocytes and embryos
Abstracts - 6th International Symposium on Preimplantation Genetics 2005
O 27 First clinical application of multiple displacement amplification in PGD Lledó B1, Ten J2, Iñiguez J2, Galán FM1, Bernabeu R3 1Molecular Biology and Genetics, Instituto Bernabeu; 2Biology of Reproduction, Instituto Bernabeu; 3Reproductive Medicine, Instituto Bernabeu, Alicante 03016, Spain Introduction: The availability of adequate quality and quantity of genomic DNA is fundamental to preimplantation genetic diagnosis (PGD). Multiple displacement amplification (MDA) is a new technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high quality DNA. Materials and methods: We used MDA to amplify the whole genome directly from a single cell. MDA product was used for PCR analysis of five different loci: Huntington disease expansion, fragile X syndrome, two polymorphic repeat sequences used in Marfan syndrome (MS) diagnosis and a new X/Y chromosome marker,X22. MDA–PGD was developed for MS in a couple in which the husband is affected. Results: We report that isothermal whole genome amplification from single cell allowed analysis of five different loci. MDA– PGD developed for MS allowed diagnosis of seven embryos that were biopsed and their cells analysed. Two healthy embryos were transferred 48 h later. Conclusions: The use of MDA as a universal step marks a new era for PGD, since sufficient DNA is amplified for diagnosis of any known gene defect by standard methods and conditions.
O 28 Validating multiple displacement amplification for analysis of single cells Thornhill AR, Schowalter KV, Fredrickson JR, Walker DL, Thibodeau SN Mayo Clinic College of Medicine, Rochester, MN, USA Introduction: Multiple displacement amplification (MDA) may be used to amplify very low amounts of DNA with large yields of high-quality DNA. We validated the applicability of MDA for use on single cells as a prelude for use in clinical preimplantation genetic diagnosis. Materials and methods: Single fibroblasts containing multiple mutations (Phe508 +/wt, R117H +/wt in the CFTR gene; H63D, C282Y in the HFE gene) were lysed and processed for MDA. Up to 63 assays were performed on single cell MDA products using multiple diagnostic platforms including Fluorescent PCR (CFTR), lightcycler (HFE), Luminex technology (CFTR), and multiplex FPCR for 12 different CA repeats. Some MDA products were re-amplified using the same technique and up to 58 assays repeated to assess re-amplification fidelity. Results: Of 1751 loci analysed among 33 single cells, 1653 (94.4%) provided interpretable results with >90% accuracy. Total allele dropout rate was 18.7% (range 4–89%). Of re-amplified samples, accuracy and reliability was equivalent to original amplification. Conclusions: MDA is a simple method achieving accurate, reliable results from single cells.
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O 29 Microarray technology to assess gene expression profiles of human oocytes and embryos Steuerwald N1,3, Bermúdez MG*2, Wells D1, Munné S2, Cohen J1,4 1ART Institute of NY and NJ, 101 Old Short Hills Road, West Orange, NJ 07052; 2Reprogenetics, 101 Old Short Hills Road, West Orange, NJ 0705; 3Department of Biology, University of North Carolina, Charlotte, NC 28223; 4Tyho-Galileo Research Laboratories, PO Box 237045 Ansonia Station, NY 10023, USA Introduction: Current knowledge of molecular mechanisms during early development is fairly rudimentary. Further characterization of these mechanisms governing development in early stages in oocytes and embryos is needed to understand gene activity and the response to external factors in the ovary and reproductive tract. Materials and methods: Non-viable human oocytes and embryos were obtained from patients undergoing IVF. As such, this material is potentially compromised. Isolated RNA was linearly amplified, labelled and fragmented, finally being applied to Affymetrix GeneChip expression probe microarrays. Results: Subtle differences in expression were evident among oocytes of the same developmental stage but derived from different patient subpopulations, probably due to genetic differences that contribute to the patient’s phenotype. Comparison of embryos at different stages of development revealed large divergences of gene expression. In this case, differentially expressed genes are predicted to be responsible for orchestrating the progression through the different stages of preimplantation development. Conclusions: The orderly succession of events during oocyte maturation and embryonic development belies the complex interactions coordinated by the genes being expressed and/or silenced. Variations in expression observed between oocytes of diverse developmental competence is indicative of the functional differences. These differences in expression may form the basis of future methodologies to predict enhanced developmental prognosis and additional insights into the regulation of oocyte maturation and preimplantation development, thus providing potential targets for diagnosis and infertility treatment.
O 30 Genetic background affects gene expression profiles of mouse blastocysts Katz-Jaffe MG, Linck DW, Schoolcraft WB, Gardner DK Colorado Centre for Reproductive Medicine, CO, USA Introduction: Genetic background has been acknowledged as one of several variables that may contribute to the success of a human IVF cycle. The aim of this study was to investigate gene expression profiles of in-vivo- and in-vitro-developed blastocysts from inbred and outbred mouse strains. Materials and methods: Embryos were collected from inbred F1 and outbred CF1 female mice following superovulation at 22 h post-human chorionic gonadotrophin (HCG) for zygotes and 94 h post-HCG for in-vivo-developed blastocysts. Total RNA was extracted from groups of morphologically similar
O 27 First clinical application of multiple displacement amplification in PGD Lledó B1, Ten J2, Iñiguez J2, Galán FM1, Bernabeu R3 1Molecular Biology and Genetics, Instituto Bernabeu; 2Biology of Reproduction, Instituto Bernabeu; 3Reproductive Medicine, Instituto Bernabeu, Alicante 03016, Spain Introduction: The availability of adequate quality and quantity of genomic DNA is fundamental to preimplantation genetic diagnosis (PGD). Multiple displacement amplification (MDA) is a new technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high quality DNA. Materials and methods: We used MDA to amplify the whole genome directly from a single cell. MDA product was used for PCR analysis of five different loci: Huntington disease expansion, fragile X syndrome, two polymorphic repeat sequences used in Marfan syndrome (MS) diagnosis and a new X/Y chromosome marker,X22. MDA–PGD was developed for MS in a couple in which the husband is affected. Results: We report that isothermal whole genome amplification from single cell allowed analysis of five different loci. MDA– PGD developed for MS allowed diagnosis of seven embryos that were biopsed and their cells analysed. Two healthy embryos were transferred 48 h later. Conclusions: The use of MDA as a universal step marks a new era for PGD, since sufficient DNA is amplified for diagnosis of any known gene defect by standard methods and conditions.
O 28 Validating multiple displacement amplification for analysis of single cells Thornhill AR, Schowalter KV, Fredrickson JR, Walker DL, Thibodeau SN Mayo Clinic College of Medicine, Rochester, MN, USA Introduction: Multiple displacement amplification (MDA) may be used to amplify very low amounts of DNA with large yields of high-quality DNA. We validated the applicability of MDA for use on single cells as a prelude for use in clinical preimplantation genetic diagnosis. Materials and methods: Single fibroblasts containing multiple mutations (Phe508 +/wt, R117H +/wt in the CFTR gene; H63D, C282Y in the HFE gene) were lysed and processed for MDA. Up to 63 assays were performed on single cell MDA products using multiple diagnostic platforms including Fluorescent PCR (CFTR), lightcycler (HFE), Luminex technology (CFTR), and multiplex FPCR for 12 different CA repeats. Some MDA products were re-amplified using the same technique and up to 58 assays repeated to assess re-amplification fidelity. Results: Of 1751 loci analysed among 33 single cells, 1653 (94.4%) provided interpretable results with >90% accuracy. Total allele dropout rate was 18.7% (range 4–89%). Of re-amplified samples, accuracy and reliability was equivalent to original amplification. Conclusions: MDA is a simple method achieving accurate, reliable results from single cells.
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O 29 Microarray technology to assess gene expression profiles of human oocytes and embryos Steuerwald N1,3, Bermúdez MG*2, Wells D1, Munné S2, Cohen J1,4 1ART Institute of NY and NJ, 101 Old Short Hills Road, West Orange, NJ 07052; 2Reprogenetics, 101 Old Short Hills Road, West Orange, NJ 0705; 3Department of Biology, University of North Carolina, Charlotte, NC 28223; 4Tyho-Galileo Research Laboratories, PO Box 237045 Ansonia Station, NY 10023, USA Introduction: Current knowledge of molecular mechanisms during early development is fairly rudimentary. Further characterization of these mechanisms governing development in early stages in oocytes and embryos is needed to understand gene activity and the response to external factors in the ovary and reproductive tract. Materials and methods: Non-viable human oocytes and embryos were obtained from patients undergoing IVF. As such, this material is potentially compromised. Isolated RNA was linearly amplified, labelled and fragmented, finally being applied to Affymetrix GeneChip expression probe microarrays. Results: Subtle differences in expression were evident among oocytes of the same developmental stage but derived from different patient subpopulations, probably due to genetic differences that contribute to the patient’s phenotype. Comparison of embryos at different stages of development revealed large divergences of gene expression. In this case, differentially expressed genes are predicted to be responsible for orchestrating the progression through the different stages of preimplantation development. Conclusions: The orderly succession of events during oocyte maturation and embryonic development belies the complex interactions coordinated by the genes being expressed and/or silenced. Variations in expression observed between oocytes of diverse developmental competence is indicative of the functional differences. These differences in expression may form the basis of future methodologies to predict enhanced developmental prognosis and additional insights into the regulation of oocyte maturation and preimplantation development, thus providing potential targets for diagnosis and infertility treatment.
O 30 Genetic background affects gene expression profiles of mouse blastocysts Katz-Jaffe MG, Linck DW, Schoolcraft WB, Gardner DK Colorado Centre for Reproductive Medicine, CO, USA Introduction: Genetic background has been acknowledged as one of several variables that may contribute to the success of a human IVF cycle. The aim of this study was to investigate gene expression profiles of in-vivo- and in-vitro-developed blastocysts from inbred and outbred mouse strains. Materials and methods: Embryos were collected from inbred F1 and outbred CF1 female mice following superovulation at 22 h post-human chorionic gonadotrophin (HCG) for zygotes and 94 h post-HCG for in-vivo-developed blastocysts. Total RNA was extracted from groups of morphologically similar